Method, Instruments,and kit for autologous tansplantation

ABSTRACT

An implantable article for cartilage repair by implantation in an animal. The implantable article includes a support matrix, and chondrocyte cells and a bio-compatible adhesive adhered to an edge of the support matrix, wherein the support matrix is absorbable by the animal. The support matrix is a biocompatible material such as collagen, and the adhesive is autologous fibrin.

STATEMENT OF RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/690,252, filed Oct. 17, 2000, which is acontinuation of application Ser. No. 09/320,246, filed May 26, 1999 (nowU.S. Pat. No. 6,283,980), which in turn is a continuation of U.S. patentapplication Ser. No. 08/857,090, filed May 15, 1997 (now U.S. Pat. No.5,989,269), which is a continuation-in-part of U.S. patent applicationSer. No. 08/704,891, filed Aug. 30, 1996 (now U.S. Pat. No. 5,759,190),all of which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] The instant invention concerns the field of chondrocytetransplantation, bone and cartilage grafting, healing, joint repair andthe prevention of arthritic pathologies. In particular methods for thepreparation of the graft site, instruments for such preparation and forthe autologous transplantation of cells to the prepared graft site.

BACKGROUND OF THE INVENTION

[0003] More than 500,000 arthroplastic procedures and total jointreplacements are performed each year in the United States. Approximatelythe same number of similar procedures are performed in Europe. Includedin these numbers are about 90,000 total-knee replacements and around50,000 procedures to repair defects in the knee per year in Europe. Thenumber of procedures are essentially the same in the U.S. (In: PraemerA., Furner S., Rice, D. P., Musculoskeletal conditions in the UnitedStates, American Academy of Orthopaedic Surgeons, Park Ridge, Ill.,1992, 125). A method for regeneration-treatment of cartilage would bemost useful, and could be performed at an earlier stage of joint damage,thus reducing the number of patients needing artificial jointreplacement surgery. With such preventative methods of treatment, thenumber of patients developing osteoarthritis would also decrease.

[0004] Techniques used for resurfacing the cartilage structure in jointshave mainly attempted to induce the repair of cartilage usingsubchondral drilling, abrasion and other methods whereby there isexcision of diseased cartilage and subchondral bone, leavingvascularized cancellous bone exposed (Insall, J., Clin. Orthop.1974,101,61; Ficat R. P. et al, Clin Orthop. 1979, 144, 74; Johnson L.L., In: Operative Arthroscopy, McGinty J. B., Ed., Raven Press, NewYork, 1991, 341).

[0005] Coon and Cahn (Science 1966, 153, 1116) described a technique forthe cultivation of cartilage synthesizing cells from chick embryosomites. Later Cahn and Lasher (PNAS USA 1967, 58, 1131) used the systemfor analysis of the involvement of DNA synthesis as a prerequisite forcartilage differentiation. Chondrocytes respond to both EFG and FGF bygrowth (Gospodarowicz and Mescher, J. Cell Physiology 1977, 93, 117),but ultimately lose their differentiated function (Benya et al., Cell1978, 15, 1313). Methods for growing chondrocytes were described and areprincipally being used with minor adjustments by Brittberg, M. et al.(New Engl. J. Med. 1994, 331, 889). Cells grown using these methods wereused as autologous transplants into knee joints of patients.Additionally, Kolettas et al. (J. Cell Science 1995, 108, 1991) examinedthe expression of cartilage-specific molecules such as collagens andproteoglycans under prolonged cell culturing. They found that despitemorphological changes during culturing in monolayer cultures (Aulthouse,A. et al., In Vitro Cell Dev. Biol., 1989,25,659; Archer, C. et al., J.Cell Sci. 1990,97,361; Hanselmann, H. et al., J. Cell Sci. 1994,107,17;Bonaventure, J. et al., Exp. Cell Res. 1994,212,97), when compared tosuspension cultures grown over agarose gels, alginate beads or asspinner cultures (retaining a round cell morphology) tested by variousscientists did not change the chondrocyte—expressed markers such astypes II and IX collagens and the large aggregating proteoglycans,aggrecan, versican and link protein did not change (Kolettas, E. et al.,J. Cell Science 1995,108,1991).

[0006] The articular chondrocytes are specialized mesenchymal derivedcells found exclusively in cartilage. Cartilage is an avascular tissuewhose physical properties depend on the extracellular matrix produced bythe chondrocytes. During endochondral ossification chondrocytes undergoa maturation leading to cellular hypertrophy, characterized by the onsetof expression of type X collagen (Upholt, W. B. and Olsen, R. R., In:Cartilage Molecular Aspects (Hall, B & Newman, S, Eds.) CRC Boca Raton1991, 43; Reichenberger, E. et al., Dev. Biol. 1991, 148, 562; Kirsch,T. et al., Differentiation, 1992, 52, 89; Stephens, M. et al., J. CellSci. 1993,103,1111).

[0007] Excessive degradation of type II collagen in the outer layers orarticular surfaces of joints is also caused by osteoarthritis. Thecollagen network is accordingly weakened and subsequently developsfibrillation whereby matrix substances such as proteoglycans are lostand eventually displaced entirely. Such fibrillation of weakenedosteoarthritic cartilage can reach down to the calcified cartilage andinto the subchondral bone (Kempson, G. E. et al., Biochim. Biophys. Acta1976, 428, 741; Roth, V. and Mow, V. C., J. Bone Joint Surgery, 1980,62A, 1102; Woo, S. L.-Y. et al., in Handbook of Bioengineering (R.Skalak and S. Chien eds.), McGraw-Hill, New York, 1987, pp. 4.1-4.44).

[0008] Descriptions of the basic development, histological andmicroscopic anatomy of bone, cartilage and other such connective tissuescan be found for example in Wheater, Burkitt and Daniels, FunctionalHistology, 2nd Edition, (Churchill Livingstone, London, 1987, Chp. 4).Descriptions of the basic histological anatomy of defects in bone,cartilage and other connective tissue can be found for example inWheater, Burkitt, Stevens and Lowe, Basic Histopathology (ChurchillLivingstone, London, 1985, Chp. 21).

[0009] Despite the advances in cultivating chondrocytes, andmanipulating bone and cartilage, there has not been great success withthe attempts to transplant cartilage or chondrocytes for the repair ofdamaged articulating surfaces. The teachings of the instant inventionprovide for effective, and efficient means of promoting thetransplantation of cartilage and/or chondrocytes into a defect in anarticulating joint or other cartilage covered bone surface, wherebycartilage is regenerated to fix the defect. The instant invention alsoprovides for surgical instruments which are designed to prepare thegraft site so as to facilitate the efficient integration of graftedmaterial to the graft site.

BRIEF SUMMARY OF THE INVENTION

[0010] The instant invention provides a method for the effectivetreatment of articulating joint surface cartilage by the transplantationof chondrocytes in a suitable matrix, to a surface to be treated, with ahemostatic barrier and a cell-free covering-patch comprising; firstplacing a hemostatic barrier proximal to the surface to be treated,placing chondrocytes in a suitable matrix upon the surface to be treateddistal to the hemostatic barrier, covering the surface to be treatedwith a cell-free covering-patch. A hemostatic barrier, as will befurther described below, is a barrier which inhibits the penetration ofvascularizing cells and tissue into the grafted material. In particular,the instant method provides for a hemostatic barrier that is aresorbable, semi-permeable material which inhibits or prohibits vascularinfiltration through the barrier. In one embodiment the hemostaticbarrier contains collagen. Cell-free, is used herein as in the art, andmeans a material that is substantially free from intact cells which arecapable of further cell division, promulgation or biological activity.In a preferred embodiment, a cell-free material is free from all intactnucleated cells. In one embodiment, the instant method encompasses theuse of a cell-free covering patch which contains a semi-permeablecollagen matrix. In one preferred embodiment of the method, the poroussurface of the cell-free covering-patch is directed towards the implantmaterial.

[0011] The instant invention further provides for the autologoustransplantation of collagen or chondrocytes to a graft site, wherein thegraft site has first been prepared by surgical manipulation to betteraccept the grafted material. In one embodiment, the graft site issculpted such that the walls of the graft site are contoured in anundulating pattern such that the grafted material, when placed withinthe graft site and expanded to contact the graft site wall, providesresistance against removal or expulsion of the entire graft from thegraft site. The instant invention further provides for surgicalinstruments designed to sculpt the graft site as taught by the method ofthe invention.

[0012] The invention further provides for a kit for cartilage and/orchondrocyte transplantation onto the surface of an articular jointwherein said kit comprises a hemostatic barrier, cell-freesemi-permeable covering-patch, and organic glue. In a furtherembodiment, the kit can optionally further provide one or more surgicalinstruments which can be used to sculpt the graft site in accordancewith the methods of the instant invention.

[0013] The present invention further provides an implant for cartilagerepair in an animal. In one embodiment, the implant is 1) a supportmatrix, 2) chondrocyte cells, and 3) a bio-compatible adhesive adheredto an edge of the support matrix. In one embodiment, the support isabsorbable by the animal.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present invention will be better understood by examining thefollowing figures which illustrate certain properties of the instantinvention wherein:

[0015]FIG. 1A is a drawing showing a typical articulating end of a bone.Typically, the bone material is covered on the articulating surface witha cartilaginous cap (shown by cross-hatching).

[0016]FIG. 1B shows an example of where a defect or injury to thecartilaginous cap occurs (gap in the cross-hatching), and such a defectcan be treated directly, enlarged slightly, or sculpted to accept thegrafted material by surgical procedures prior to treatment.

[0017]FIG. 1C shows how the hemostatic barrier (solid black, numbered 1)is placed within the defect in the cartilage cap to inhibit or preventvascularization into regenerating cartilage, from the underlying bone.The chondrocytes to be implanted into the defect cavity are then layeredon top of the hemostatic barrier.

[0018]FIG. 2 is a drawing showing the treated defect (gap incross-hatched area) in the cartilaginous cap (cross-hatched area)covered by a cell-free semi-permeable material (solid black, numbered 2)which is used to form a cap/patch or bandage over the defect site. Thiscap is fixed in place, either sutured to the edge of the cavity intohealthy cartilage, or otherwise attached. This cap is covering thedefective area of the joint into which the glue and culturedchondrocytes/cartilage transplant has been placed.

[0019]FIG. 3A is a diagram illustrating the differential response tocompression and shearing forces by harder and softer cartilage withsubsequent zone of demarcation.

[0020]FIG. 3B illustrates the graft site, after the defect has beensculpted to have undulating walls.

[0021]FIG. 3C illustrates the sculpted graft site with the hemostaticbarrier (1), transplanted material (3), and cell-free covering-patch (2)in place within the articular surface cartilage (4).

[0022]FIG. 4A illustrates one embodiment of the surgical device of theinstant invention showing cutting teeth (5) and protruding placement pin(6).

[0023]FIG. 4B illustrates a second embodiment of the surgical device ofthe instant invention.

[0024]FIG. 5 is a diagram illustrating the modified differentialresponse to compression and shearing forces by harder cartilage andsofter cartilage after sculpting the graft site.

[0025]FIG. 6A is an MRI image of a pig knee showing cartilage defect inleft (medial) condyle.

[0026]FIG. 6B is an MRI image of the same pig knee three months aftertreatment.

[0027]FIG. 7 illustrates a graft site or defect (23) without ahemostatic barrier, including a mixture of transplanted cells (21) andbio-compatible glue (22) on a support matrix (18) having a porous side,a cartilaginous material and transplanted material interface (30),cartilage (10), a bone material and cartilaginous material interface(12), a resorbable pin (24) and a bone material (14).

DETAILED DESCRIPTION OF THE INVENTION

[0028] This invention concerns the use of certain products that inhibitthe formation of vascular tissue, for instance such as capillary loopsprojecting into the cartilage being established, during the process ofautologous transplantation of chondrocytes into defects in thecartilage. The formation of vascular tissue from the underlying bonewill tend to project into the new cartilage to be formed leading toappearance of cells other than the mesenchymal specialized chondrocytesdesired.

[0029] The contaminating cells introduced by the vascularization maygive rise to encroachment and over-growth into the cartilage to beformed by the implanted chondrocytes. One of the types of commercialproducts which can be used in this invention is Surgicel(® (EthiconLtd., UK) which is absorbable after a period of 7-14 days. The use ofthis material in the method of the instant invention is contrary to thenormal use of a hemostatic device, such as Surgicel® as it is describedin the package insert from Ethicon Ltd.

[0030] Surprisingly, we have found that in a situation where you wish toinhibit re-vascularization into cartilage, a hemostatic material willact like a gel-like artificial coagulate. If red blood cells should bepresent within the full-thickness defect of articular cartilage that iscapped by such a hemostatic barrier, these blood cells will bechemically changed to hematin, and thus rendered unable to inducevascular growth. Thus, a hemostatic product used as a re-vascularizationinhibitory barrier with or without fibrin adhesives, such as for examplethe Surgicel®, is effective for the envisioned method as taught by theinstant invention. Another part of this invention is the use of acell-free component, that is used as a patch covering the defective areaof the joint into which the cultured chondrocytes/cartilage are beingtransplanted, using autologous chondrocytes for the transplantation. Themethod of the invention also contemplates the use of suitable allogenicchondrocytes or xenogenic chondrocytes for the repair of a cartilagedefect.

[0031] Thus the instant invention teaches methods for effective repairor treatment of cartilage defects in articular joint bone surfaces whichcomprises administering an agent or device to block vascular invasioninto the cartilage site to be repaired, and also providing for acell-free barrier which will isolate the repair site and keeptransplanted cells in place. The instant invention also provides for akit comprising a hemostatic barrier component for insertion into thesite to be repaired, such that there is effective inhibition ofvascularization into the site to be repaired; and once the chondrocytesto be transplanted are placed into the site to be repaired, a cell-freesemi-permeable barrier is capped over the repair site such that thetransplanted chondrocytes are held in place, but are still able to gainaccess to nutrients.

[0032] Certain aspects of the invention have been exemplified using anin vitro system to study the behavior of the chondrocytes when incontact with a certain product or a combination of certain products thatinhibit the formation of vascular tissue. This in vitro testing predictsthe ability of certain tested materials to inhibit vascularization, aswill occur in vivo where capillary loops project into the cartilagebeing established during the process of autologous transplantation ofchondrocytes into defects in the cartilage.

[0033] Suitable hemostatic products will be characterized by having theability to inhibit the growth, or invasion of vascular tissue,osteocytes, fibroblasts etc. into the developing cartilage. A suitablehemostatic material will achieve the goal of the method of the instantinvention in that vascular and cellular invasion into developingcartilage should be prevented in order to optimize the formation ofcartilage and achieve repair of the full-thickness of any defects in thearticular cartilage. Ideally, the hemostatic barrier will be stable foran extended period of time sufficient to allow for full cartilagerepair, and then be able to be resorbed or otherwise broken down overtime. One material identified as suitable is called Surgicel® W1912 (anabsorbable hemostat containing oxidized regenerated sterile cellulose;Lot GG3DH, Ethicon Ltd. UK). Another example of a suitable material isBioGide® (a commercially available type I collagen matrix pad; GeistlichSohne, Switzerland).

[0034] Suitable organic glue material can be found commercially, such asfor example Tisseel® or Tissucol® (fibrin based adhesive; Immuno AG,Austria), Adhesive Protein (Cat. #A-2707, Sigma Chemical, USA), and DowCorning Medical Adhesive B (Cat. #895-3, Dow Corning, USA).

[0035] The surgical instruments contemplated by the instant inventioncan be manufactured from metal and/or plastic suitable for makingsingle-use disposable, or multi-use reusable surgical instruments. Thecutting instrument may contain cutting teeth that are fully circular orflat, or anything in between. As cartilage is a relatively soft materialit may be advantageous to manufacture hardened plastic cutting edgeswhich will be able to sculpt cartilage without being able to damagebone. Such cutting instruments can be manufactured to incorporateopenings for administration of fluid, suction removal of cutting debrisand fluid, and fiber optic threads for illumination and visualization ofthe defect site.

[0036] In another embodiment, the present invention includes cells 21and glue 22 combined together as (1) a mixture of glue 22 and cells 21or (2) one or more alternating layers of cells 21 and glue 22 on supportmatrix 18.

[0037] In one such embodiment, it is contemplated that cells 21 areautologous chondrocyte cells to be transplanted into a defect 23.Chondrocytes 21 are mixed, either homogeneously or non-homogeneously,with a suitable glue 22 before application of the chondrocyte/gluemixture to support matrix 18. Preferably, glue 22 and chondrocytes 21are mixed immediately (that is, in the operating theater) beforeapplying glue 22 and cells 21 to support matrix 18, and implantation ofthe combination of glue 22, cells 21, and support matrix 18 into defect23. Alternatively cells 21 and glue 22 are alternately applied in one ormore layers to support matrix 18. In one embodiment, glue 22 is abio-compatible glue, such as a fibrin glue, and more specifically eitheran autologous fibrin glue or a non-autologous fibrin glue. Preferably,an autologous fibrin glue is used.

[0038] When chondrocytes 21 are combined with glue 22 on support matrix23, the glue/chondrocyte/support matrix combination is implanted intodefect 23 either with or without a hemostatic barrier (as describedherein) between bone 14 and the glue/chondrocyte/support matrixcombination. In one embodiment, as shown in FIG. 2, treated defect 23does not contain a hemostatic barrier between bone 14 and theglue/chondrocyte/support matrix combination.

[0039] In one embodiment, shown in FIG. 7, the treatment of defect 23according to the present invention similarly is accomplished without theuse of a hemostatic barrier between the glue/chondrocyte/support matrixcombination and the base of defect 23.

[0040] Additionally, in the embodiment shown in FIG. 7, the combinationof glue 22 and chrondrocytes 21 is present only on a portion of supportmatrix 18. For example, glue 22 and chondrocytes 21 preferably are onlyon an outer edge of a porous side of support matrix 18. Alternatively,chondrocytes 21 and/or glue 22 penetrate into a porous side of supportmatrix 18 to a desired level, for example 2 to 50% of the depth ofsupport matrix 18. Typically the porosity of support matrix 18 isgreatest nearest a rough side, i.e., the side on which cells 21 and glue22 are deposited, and least porous on the opposite side of supportmatrix 18. As further illustrated by FIG. 7, the porosity of supportmatrix 18 is indicated by shading, where the darker shading indicatesless porosity, and the lighter shading indicates greater porosity.

[0041] In yet another embodiment, chondrocytes 21 are applied to supportmatrix 18, then covered with a layer of a suitable bio-compatible glue22. Although a single layer of chondrocytes 21 on support matrix 18 anda single layer of glue 22 positioned over chondrocytes 21 is sufficientfor effective treatment of a defect, more than one alternating layer ofglue 22 and chondrocytes 21 can be used. Alternatively, a layer ofbio-compatible glue 22 can be initially deposited on support matrix 18,followed by a layer of chondrocytes 21 deposited on glue 22.

[0042] In one embodiment, support matrix 18 is a sheet-like membercapable of supporting growth of chondrocytes 21 and of providingphysical integrity to the implantable article to facilitate manipulationthereof. In an embodiment, support matrix 18 includes polypeptides orproteins such as collagen. In particular, support matrix 18 includesequine, porcine, bovine, ovine and chicken collagen. Additionally, thecollagen can be either Type I or Type II collagen. Examples ofappropriate collagen support matrices 18 include ChondraGide® andBioGide® (commercially available type I collagen matrix pads; availablefrom Geistlich Sohne, Switzerland). Additionally, support matrix 18 isreversibly deformable and has either a rough side that is porous, asdescribed above, or a smooth side, or two rough sides. Although eitherside can be porous, in one embodiment as described herein, the roughside has greater porosity relative to the smooth side. Support matrix 18and cells 21 positioned thereon can be optionally held in place in thedefect by additional bio-compatible glue 22 and/or one or morebiocompatible resorbable pins 24, as shown in FIG. 7.

[0043] In one embodiment, one or more portions of support matrix 18 iseither solid or gel-like in character.

[0044] Certain aspects of the instant invention may be better understoodas illustrated by the following examples, which are meant by way ofillustration and not limitation.

EXAMPLE 1

[0045] In order for the Surgicel® to be used according to the inventionfor preventing development of blood vessels into autologous implantedcartilage or chondrocytes, Surgicel® was first treated with a fixative,such as glutaric aldehyde. Briefly, Surgicel® was treated with 0.6%glutaric aldehyde for 1 minute, followed by several washings toeliminate glutaric aldehyde residues that may otherwise be toxic totissue. Alternatively, the Surgicel® was treated with the fibrinadhesive called Tisseel® prior to treatment with glutaric aldehyde asdescribed in Example 2. It was found that the Surgicel® fixated forinstance with a fixative such as glutaric aldehyde, washed with sterilephysiological saline (0.9%) and stored in refrigerator, does notdissolve for 1 to 2 months. Generally, Surgicel® is resorbed in a periodbetween 7 and 14 days. This time would be too short, because a longertime is needed in preventing the development of blood vessels orvascularization as such from the bone structure into the implantedcartilage before the implanted chondrocytes have grown into a solidcartilage layer getting its nutrition requirements from the neighboringcartilage. In other words sufficient inhibition of the vascularizationis needed for a longer time, such as for instance one month. Therefore,the product should not be absorbed significantly prior to that time. Onthe other hand resorption is needed eventually. Hence, the organicmaterial used as an inhibiting barrier shall have these capabilities,and it has been found that the Surgicel® treated in this manner providesthat function.

EXAMPLE 2

[0046] The Surgicel® was also coated with an organic glue, in thisexample the glue used was Tisseel® but others can also be used. Thisproduct, together with the Surgicel® produces a useable barrier for theparticular purpose of the invention. Any other hemostat or vascularinhibiting barrier could be used. The Tisseel® was mixed as describedbelow. The Surgicel® was then coated with Tisseel® by spraying theSurgicel® material on both sides until soaked. The Tisseel® (fibringlue) was then allowed to solidify at room temperature. Immediatelyprior to completed solidification, the coated Surgicel® was then placedin 0.6% glutaric aldehyde for 1 minute and then washed with sterilephysiological (0.9%) saline. The pH was then adjusted by PBS and/or withNaOH until pH was stable at 7.2 to 7.4. Afterwards the thus treatedSurgicel® was then washed in tissue culture medium such as minimumessential medium/F12 with 15 mM Hepes buffer.

[0047] As mentioned in this example we have used Tisseel® as the fibrinadhesive to coat the Surgicel®. Furthermore the fibrin adhesive or gluemay also be applied directly on the bottom of the lesion towards thebone, on which the Surgicel® is glued. The in vitro system used, in lieuof in vivo testing, consisted of a NUNCLON™ Delta 6-well steriledisposable plate for cell research work (NUNC. InterMed, Roskilde,Denmark). Each well measures approximately 4 cm in diameter.

[0048] In the invention the fibrin adhesive can be any adhesive whichtogether with the fibrin component will produce a glue that can betolerated in humans (Ihara, N, et al., Burns Incl. Therm. Inj., 1984,10, 396). The invention also anticipates any other glue component thatcan be used in lieu of the fibrin adhesive. In this invention we usedTisseel® or Tissucol® (Immuno AG, Vienna, Austria). The Tisseel® kitconsists of the following components:

[0049] Tisseel®, a lyophilized, virus-inactivated Sealer, containingclottable protein, thereof: fibrinogen, Plasma fibronectin (CIG) andFactor XIII, and Plasminogen;

[0050] Aprotinin Solution (bovine);

[0051] Thrombin 4 (bovine);

[0052] Thrombin 500 (bovine); and

[0053] Calcium Chloride solution.

[0054] The Tisseel® kit contains a DUPLOJECT® Application System. Thefibrin adhesive or the two-component sealant using Tisseel® Kit iscombined in the following manner according to the Immuno AG productinsert sheet.

EXAMPLE 3

[0055] Chondrocytes were grown in minimal essential culture mediumcontaining HAM F12 and 15 mM Hepes buffer and 5 to 7.5% autologous serumin a CO₂ incubator at 37° C. and handled in a Class 100 laboratory atVerigen Europe A/S, Symbion Science Park, Copenhagen, Denmark. Othercompositions of culture medium may be used for culturing thechondrocytes. The cells were trypsinized using trypsin EDTA for 5 to 10minutes and counted using Trypan Blue viability staining in aBurker-Turk chamber. The cell count was adjusted to 7.5×10⁵ cells perml. One NUNCLON™ plate was uncovered in the Class 100 laboratory.

[0056] The Surgicel® hemostatic barrier was cut to a suitable sizefitting into the bottom of the well in the NUNCLON™ tissue culture tray.In this case a circle, of a size of approximately 4 cm (but could be ofany possible size) and placed under aseptic conditions on the bottom inwell in a NUNCLON™ Delta 6-well sterile disposable plate for cellresearch work (NUNC, InterMed, Roskilde, Denmark). The hemostaticbarrier to be placed on the bottom of the well was pre-treated asdescribed in Example 1. This treatment delays the absorption of theSurgicel significantly. This hemostatic barrier was then washed severaltimes in distilled water and subsequently several times untilnon-reacted glutaraldehyde was washed out. A small amount of the cellculture medium containing serum was applied to be absorbed into thehemostatic barrier and at the same time keeping the hemostatic barrierwet at the bottom of the well.

[0057] Approximately 10⁶ cells in 1 ml culture medium were placeddirectly on top of the hemostatic barrier, dispersed over the surface ofthe hemostatic barrier pre-treated with 0.4% glutaraldehyde as describedabove. The plate was then incubated in a CO₂ incubator at 37° C. for 60minutes. An amount of 2 to 5 ml of tissue culture medium containing 5 to7.5% serum was carefully added to the well containing the cells avoidingsplashing the cells by holding the pipette tip tangential to the side ofthe well when expelling the medium. It appeared that the pH of themedium was too low (pH˜6.8). The pH was then adjusted to 7.4 to 7.5. Thenext day some chondrocytes had started to grow on the hemostaticbarrier, arranged in clusters. Some of the cells had died due to the lowpH exposure prior to the adjustment of the pH. The plate was incubatedfor 3 to 7 days with medium change at day 3.

[0058] At the end of the incubation period the medium was decanted, andcold refrigerated 2.5% glutaraldehyde containing 0.1 M sodium salt ofdimethylarsinic acid, (also called sodium cacodylate, pH is adjustedwith HCl to 7.4), was added as a fixative for preparation of the celland supporter (hemostatic barrier) for later preparation for electronmicroscopy.

EXAMPLE 4

[0059] Chondrocytes were grown in minimal essential culture mediumcontaining HAM F12 and 15 mM Hepes buffer and 5 to 7.5% autologous serumin a CO₂ incubator at 37° C. and handled in a Class 100 laboratory atVerigen Europe A/S, Symbion Science Park, Copenhagen, Denmark. Othercompositions of culture medium may be used for culturing thechondrocytes. The cells were trypsinized using trypsin EDTA for 5 to 10minutes and counted using Trypan Blue viability staining in aBurker-Turk chamber. The cell count was adjusted to 7.5×10⁵ cells perml. One NUNCLON™ plate was uncovered in the Class 100 laboratory.

[0060] The Surgicel® (for use as the hemostatic barrier) was treatedwith 0.6% glutaric aldehyde for one minute as described in Example 1,and washed with 0.9% sterile sodium chloride solution or, preferably,with a buffer such as a PBS buffer or the culture medium such asMEM/F12, because the pH after the glutaric aldehyde treatment is 6.8 andshould preferably be 7.0 to 7.5. The Tisseel® was applied on both sidesof the Surgicel® using the DUPLOJECT® system, thus coating both sides ofthe Surgicel®, the patch intended to be used, with fibrin adhesive. Theglue is left to dry under aseptic condition for at least 3 to 5 minutes.The “coated” hemostatic barrier was placed on the bottom of the well ina NUNCLON™ Delta 6-well sterile disposable plate for cell research work.A small amount of tissue culture medium containing serum was applied tobe absorbed into the hemostatic barrier. Approximately 10⁶ cells in 1 mltissue culture medium containing serum was placed directly on top of theHemostat, dispersed over the surface of the hemostatic barrier. Theplate was then incubated in a CO₂ incubator at 37° C. for 60 minutes. Anamount of 2 to 5 ml of tissue culture medium containing 5 to 7.5% serumwas carefully added to the well containing the cells avoiding splashingthe cells by holding the pipette tip tangential to the side of the wellwhen expelling the medium. After 3 to 6 days, microscopic examinationshowed that the cells were adhering to and growing into the Surgicel® ina satisfactory way suggesting that Surgicel® did not show toxicity tothe chondrocytes and that the chondrocytes grew in a satisfactory mannerinto the Surgicel®.

[0061] The plate was incubated for 3 to 7 days with medium change at day3. At the end of the incubation period the medium was decanted, and coldrefrigerated, and 2.5% glutaraldehyde containing 0.1 M sodium salt ofdimethylarsinic acid, also called sodium cacodylate, pH is adjusted withHCl to 7.4, was added as a fixative for preparation of the cell andsupporter (hemostatic barrier) for later preparation for electronmicroscopy.

EXAMPLE 5

[0062] Chondrocytes were grown in minimal essential culture mediumcontaining HAM F12 and 15 mM Hepes buffer and 5 to 7.5% autologous serumin a CO₂ incubator at 37° C. and handled in a Class 100 laboratory atVerigen Europe A/S, Symbion Science Park, Copenhagen, Denmark. The cellswere trypsinized using trypsin EDTA for 5 to 10 minutes and countedusing Trypan Blue viability staining in a Burker-Turk chamber. The cellcount was adjusted to 7.5×10⁵ to 2×10⁶ cells per ml. One NUNCLON™ platewas uncovered in the Class 100 laboratory.

[0063] It has been found that the Bio-Gide® can be used as a resorbablebilayer membrane which will be used as the patch or bandage covering thedefective area of the joint into which the cultured chondrocytes arebeing transplanted as well as the hemostatic barrier. The Bio-Gide® is apure collagen membrane obtained by standardized, controlledmanufacturing processes (by E. D. Geistlich Sohne AG, CH-6110 Wolhusen).The collagen is extracted from veterinary certified pigs and iscarefully purified to avoid antigenic reactions, and sterilized indouble blisters by gamma irradiation. The bilayer membrane has a poroussurface and a dense surface. The membrane is made of collagen type I andtype III without further cross-linking or chemical treatment. Thecollagen is resorbed within 24 weeks. The membrane retains itsstructural integrity even when wet and it can be fixed by sutures ornails. The membrane may also be “glued” using fibrin adhesive such asTisseel® to the neighboring cartilage or tissue either instead ofsutures or together with sutures.

[0064] The Bio-Gide® was un-covered in a class 100 laboratory and placedunder aseptic conditions on the bottom of the wells in a NUNCLON™ Delta6-well sterile disposable plate for cell research work,—either with theporous surface of the bilayer membrane facing up or with the densesurface facing up. Approximately 10⁶ cells in 1 ml tissue culture mediumcontaining serum was placed directly on top of the Bio-Gide®, dispersedeither over the porous or the dense surface of the Bio-Gide®. The platewas then incubated in a CO₂ incubator at 37° C. for 60 minutes. Anamount of 2 to 5 ml of tissue culture medium containing 5 to 7.5% serumwas carefully added to the well containing the cells avoiding splashingthe cells by holding the pipette tip tangential to the side of the wellwhen expelling the medium.

[0065] On day 2 after the chondrocytes were placed in the wellcontaining the Bio-Gide® the cells were examined in a Nikon Invertedmicroscope. It was noticed that some chondrocytes had adhered to theedge of the Bio-Gide. It was of course not possible to look through theBio-Gide® itself using this microscope.

[0066] The plate was incubated for 3 to 7 days with medium change at day3. At the end of the incubation period the medium was decanted, and coldrefrigerated 2.5% glutaraldehyde containing 0.1 M sodium salt ofdimethylarsinic acid, also called sodium cacodylate, pH is adjusted withHCl to 7.4, was added as fixative for preparation of the cell and theBio-Gide® supporter with the cells either cultured on the porous surfaceor the dense surface. The Bio-Gide® patches were then sent for electronmicroscopy at Department of Pathology, Herlev Hospital, Denmark.

[0067] The electron microscopy showed that the chondrocytes cultured onthe dense surface of the Bio-Gide® did not grow into the collagenstructure of the Bio-Gide®, whereas the cells cultured on the poroussurface did indeed grow into the collagen structure and furthermore,showed presence of proteoglycans and no signs of fibroblast structures.This result shows that when the collagen patch, as for instance aBio-Gide® patch is sewn as a patch covering a cartilage defect theporous surface shall be facing down towards the defect in which thecultured chondrocytes are to be injected. They will then be able topenetrate the collagen and produce a smooth cartilage surface in linewith the intact surface, and in this area a smooth layer ofproteoglycans will be built up. Whereas, if the dense surface of thecollagen is facing down into the defect the chondrocytes to be implantedwill not integrate with the collagen, and the cells will not produce thesame smooth surface as described above.

EXAMPLE 6

[0068] Chondrocytes were grown in minimal essential culture mediumcontaining HAM F12 and 15 mM Hepes buffer and 5 to 7.5% autologous serumin a CO₂ incubator at 37° C. and handled in a Class 100 laboratory atVerigen Europe A/S, Symbion Science Park, Copenhagen, Denmark. The cellswere trypsinized using trypsin EDTA for 5 to 10 minutes and countedusing Trypan Blue viability staining in a Burker-Turk chamber. The cellcount was adjusted to 7.5×10⁶ to 2×10⁶ cells per ml. One NUNCLON™ platewas uncovered in the Class 100 laboratory.

[0069] The Bio-Gide® used as a resorbable bilayer membrane may also beused together with an organic glue such as Tisseel® with additional,significantly higher content of Aprotinin than normally found inTisseel®, as described in the product insert. By increasing the contentof Aprotinin to about 25,000 KIU/ml, the resorption of the material willbe delayed by weeks instead of the normal span of days.

[0070] To test this feature in vitro, the Tisseel® is applied to thebottom of the well of the NUNCLON™ plate, and allowed to solidifyincompletely. A collagen patch such as a Bio-Gide® is then applied overthe Tisseel® and glued to the bottom of the well. This combination ofBio-Gide® and Tisseel® is designed to be a hemostatic barrier that willinhibit or prevent development or infiltration of blood vessels into thechondrocyte transplantation area. This hybrid collagen patch can now beused for both a hemostatic barrier at the bottom of the lesion (mostproximal to the surface to be repaired) as well as a support forcartilage formation because the distal surface can be the porous side ofthe collagen patch and thus encourage infiltration of chondrocytes andcartilage matrix. Thus, this hybrid collagen patch can also be used tocover the top of the implant with the collagen porous surface directeddown towards the implanted chondrocytes and the barrier forming the top.The hybrid collagen patch, with elevated Aprotinin component may also beused without any organic glue such as Tisseel® and placed within thedefect directly, adhering by natural forces. Thus the collagen patch canbe used both as the hemostatic barrier, and the cell-free covering ofthe repair/transplant site, with the porous surfaces of the patchesoriented towards the transplanted chondrocytes/cartilage. Anothervariant would use a collagen patch which consists of type II collagen(i.e. from Geistlich Sohne AG, CH-6110 Wolhusen).

[0071] Thus the instant invention provides for a hybrid collagen patchwhere said patch is a collagen matrix with elevated levels of aprotinincomponent, preferably about 25,000 KIU/ml, in association with anorganic matrix glue, where the collagen component is similar to theBio-Gide® resorbable bilayer material or Type II collagen, and theorganic glue is similar to the Tisseel® material. In another embodiment,the hybrid collagen patch does not use any organic glue to adhere to thesite of the repair.

EXAMPLE 7

[0072] Because of the weakened structure of osteoarthritic cartilage,adherence of cultured autologous chondrocytes transplanted to a graftsite in defective cartilage may be inhibited, thus creating a marginalzone (zone of demarcation) between the newly implantedcartilage/chondrocytes and the surrounding established cartilage. Thismarginal zone will be most pronounced if the graft site is prepared forthe graft by creating straight, smooth walls cut in a linear fashion.The shearing and compression forces across such a marginal zone (asillustrated in FIG. 3A) will exert great force to dislodge the graftwhen the graft site is cut in a linear fashion. This marginal zone, anddifferential movement of materials along this zone will inhibitconfluent healing between the grafted material and the surroundingmaterial. In many cases the graft material is softer than thesurrounding material, however, in some instances of osteoarthritisdisease, the surrounding cartilage may in fact be softer than theimplanted chondrocytes/cartilage.

[0073] Therefore, in order to solve this problem, the method of theinvention teaches the use of surgical instruments to sculpt the walls ofthe graft site such that the walls are non-linear, and thus provide forundulated surfaces. It is also possible to shape the graft site suchthat the diameter of the site proximal to the bone surface is of agreater dimension then the opening distal to the bone, and at thesurface of the cartilage. However, the preferred embodiment describesthe sculpting of the walls of the graft site in an fashion similar to athreaded opening for receiving a bolt or screw (as illustrated in FIG.3B), thus providing mechanical resistance to the compression and orejection of the grafted material from the graft site which can bedescribed as “male” and “female” threading.

[0074] The surgical instruments contemplated by the instant inventioncan be manufactured from metal and/or plastic suitable for makingsingle-use disposable, or multi-use reusable surgical instruments. Ascartilage is a relatively soft material it may be advantageous tomanufacture hardened plastic cutting edges which will be able to sculptcartilage without being able to damage bone. Such cutting instrumentscan be manufactured to incorporate openings for administration of fluid,suction removal of cutting debris and fluid, and fiber optic threads forillumination and visualization of the defect site. In certainembodiments of the instrument, the base of the instrument may haveprotruding point or pin-like structure which will assist in guiding andplacing the instrument in the graft site. Of course such a pin would bedesigned to minimized damage to the underlying bone.

[0075] While the cutting surface of the instrument may be singletoothed, or multi-toothed, or describe a screw-like pattern such as thatin a metal tap used to generate threaded holes in metal parts, thecharacteristic required of the cutting instrument is that the resultingsculpted sides of the graft site is undulated, and non-linear. Forexample, in certain embodiments, the cutting edge of the instrument canbe shaped similar to that shown in FIG. 4A, or as in FIG. 4B. Thecutting edge maybe flat, or circular in that it wraps around thediameter of the cutting instrument. Many other shapes can be designed toaccomplish the purpose of the method of the invention to create aninterface which provides for mechanical resistance to differentialreaction to compression and shearing forces on the transplanted materialand the surrounding material.

EXAMPLE 8

[0076] A four month old mixed Yorkshire breed pig was subjected togeneral anesthesia and placed on its back. The pig was washed and drapedin a surgical suite at Harrington Arthritis Research Center, Phoenix,Ariz. The entire surgical procedure was performed aseptically. The lefthind-leg and adjacent abdomen and inguinal area was cleaned with iodine.The knee joint was localized, and the patella localized. A medialincision was performed approximately 3 cm from the posterior part of thepatella and the several subcutis, muscle layers and ligaments was cutapproximately in order to get access to the medial femoral condyle.Using a circular cutter a lesion was prepared in the white cartilage onthe medial part of the medial condyle, leaving a 0.5 to 1 cm margin tothe edge of the cartilage covering the posterior-medial part of thecondyle (left condyle, FIG. 6A). The 0.5 to 1 cm defect was placed in acaudal weight bearing part of the medial condyle. The entire surgicalprocedure was done without tourniquet on the left femur. The differentlayers and skin was sutured appropriately.

[0077] On day 3 the animal was again brought to the surgical suite andpositioned as above on the operating table and given general anesthesia.The left hind leg, abdomen and inguinal region was cleaned with iodine,as described above. Sutures were cut and the area opened. It was noticedthat a moderate hematoma was present in the knee joint. The blood clotwas removed and the defect inspected. There was a blood clot in thedefect which was removed. A sterile surgical instrument designed with amale thread cutting edge, with a size corresponding to, or slightlybigger than the circumference of the lesion was carefully screwed downinto the defect. A BioGide® pad was cut to a size equal to the bottom ofthe defect. The first glue used, called Adhesive Protein (A-2707, SigmaChemical, USA) was applied on the dense side of the trimmed hemostaticbarrier pad, and the pad was placed dense side down into the bottom ofthe lesion, using it as a barrier as described above. It was found thatthis glue did not seem to dry very fast. The slight bleeding from thebottom of the defect stopped immediately. A second BioGide® was cutsomewhat bigger in circumference than the lesion and was placed withdense side up (thus the porous side down towards the graft) as describedabove.

[0078] This non-cellular covering-pad was then sutured over the cavity,leaving one edge open, where the chondrocyte to be explanted could beinjected. The surrounding part of the edge of the pad was covered withthe second glue, Dow Corning Medical Adhesive B (Cat. #895-3, DowCorning, USA). This second glue dried much faster and more efficientlythan the first glue. It was found that during this particular procedure,the first glue had not dried sufficiently to hold the hemostatic barrierin place when suturing of the cap was attempted. Thus, the main barrierformed on the proximal surface of the graft site was by the glue itself.

[0079] Using a 1 ml syringe and a 16 gauge needle, the chondrocyte cellsuspension (about 0.6 ml) was drawn up into the barrel of the syringe. A23 gauge short needle was switched for the 16 gauge needle, and the cellsuspension was injected under the sutured covering-patch into the graftsite (about 10×10⁶ cells). The open edge of the cap was then glued priorto removal of the needle, and the needle carefully withdrawn. No leakageof cells was seen. The wound was sutured and as above, no tourniquet wasused, no bleeding was observed. The final skin layers were sutured. Noprotrusion of the skin occurred after suturing, which indicates thatthere was no hematoma. Postoperative recovery was uneventful.

[0080] As expected, the grafted chondrocytes produced cartilage matrixsufficient to repair the defect made in the articular cartilage surfaceof the knee joint of the test pig. FIG. 6A is an MRI image of a pig kneeshowing the cartilage defect created in the knee (left condyle, themedial condyle), and FIG. 6B is an MRI image of the same pig knee threemonths after treatment showing repair of the defect.

EXAMPLE 9

[0081] A kit comprising the components useful for practicing the methodof the invention, will allow for the convenient practice of the methodof the invention in a surgical setting. In a preferred embodiment, a kitof the invention will provide sterile components suitable for easy usein the surgical environment, and will provide a suitable hemostaticbarrier, suitable covering patch, and if needed organic glue. A kit ofthe invention may also provide sterile, cell-free matrix materialsuitable for supporting autologous chondrocytes that are to be implantedinto an articular joint surface defect. In one embodiment, a kit of theinvention contains a Surgicel® hemostatic barrier and a Bio-Gide®covering patch with suitable coating of Tisseel® organic glue, where theSurgicel® and Bio-Gide® have been treated according to the teachings ofthe invention to increase the time till resorption. In instances whereTisseel® is pre-coated, in one embodiment the Tisseel® is supplementedwith additional aprotinin to increase time till resorption.

[0082] In another preferred embodiment, the hemostatic barrier andcovering-patch are both a semi-permeable collagen matrix which istreated to extend the time till resorption of the material. It is alsopossible to provide Tisseel® glue in enhanced form as a separatecomponent to be applied as needed because of the inherent variabilityand unique circumstances every repair/transplantation procedure willencounter.

[0083] A further embodiment of the kit will include a surgicalinstrument as described in Example 7 above.

[0084] It will be appreciated by persons skilled in the art thatnumerous variations and/or modifications may be made to the inventionshown in the specific embodiments without departing form the spirit andscope of the invention as broadly described.

What is claimed is:
 1. An implantable article for cartilage repair byimplantation in an animal comprising a support matrix, and chondrocytecells and a bio-compatible adhesive adhered to an edge of said supportmatrix, wherein said support matrix is absorbable by the animal.
 2. Theimplantable article of claim 1, wherein said support matrix is asheet-like member capable of supporting growth of the chondrocyte cellsand of providing physical integrity to the implantable article tofacilitate manipulation thereof.
 3. The implantable article of claim 1,wherein said support matrix comprises polypeptides or proteins.
 4. Theimplantable article of claim 1, wherein said support matrix is collagen.5. The implantable article of claim 1, wherein said collagen is selectedfrom the group consisting essentially of equine, porcine, bovine, ovineand chicken collagen.
 6. The implantable article of claim 1, whereinsaid support matrix is solid.
 7. The implantable article of claim 1,wherein said support matrix is gel-like.
 8. The implantable article ofclaim 4, wherein said collagen is collagen Type I.
 9. The implantablearticle of claim 4, wherein said collagen is collagen Type II.
 10. Theimplantable article of claim 1, wherein said implantable article isreversibly deformable.
 11. The implantable article of claim 1, whereinsaid support matrix has a rough side.
 12. The implantable article ofclaim 11, wherein said rough side is porous.
 13. The implantable articleof claim 1, wherein said support matrix has a smooth side.
 14. Theimplantable article of claim 1, wherein said support matrix has a roughand a smooth side.
 15. The implantable article of claim 1, wherein saidchondrocyte cells and said bio-compatible adhesive are in an admixture.